DNA-MEDIATED FOLDING AND ASSEMBLY OF MYOD-E47 HETERODIMERS

Basic region helix-loop-helix (bHLH) transcription factors regulate ke y steps in early development by binding to regulatory DNA sites as het erodimers consisting of a tissue-specific factor and a widely expresse d factor. We have examined the folding, dimerization, and DNA binding properties of the muscle-specific bHLH protein MyoD and its partner E4 7, to understand why these proteins preferentially associate in hetero dimeric complexes with DNA. In the absence of DNA, the E47 bHLH domain forms a very stable homodimer, whereas MyoD is unfolded and monomeric . Fluorescence quenching experiments show that MyoD does not dimerize with E47 under dilute conditions in the absence of DNA. Residues in an d around the loop of the E47 bHLH domain contribute to its markedly gr eater stability. An altered MyoD bHLH substituted with the loop segmen t from E47 folds in the absence of DNA, and it readily dimerizes with E47. In the presence of a specific DNA binding site, MyoD and E47 both form homodimeric complexes with DNA that have similar dissociation co nstants, despite the very different stabilities of these protein dimer s off DNA. A 1:1 mixture of these bHLH domains forms almost exclusivel y heterodimeric complexes on DNA. Assembly of these bHLH-DNA complexes is apparently governed by the strength of each subunit's interaction with the DNA and not by the strength of protein-protein interactions a t the dimer interface. These findings suggest that preferential associ ation of MyoD with E47 in DNA complexes results from more favorable DN A contacts made by one or both subunits of the heterodimer in comparis on with either homodimeric complex.